The most important factor for cancer development in the United States is exposure to tobacco products. The most prevalent route of exposure to tobacco is cigarette smoking with increasing use of smokeless tobacco products in recent years. Among the most important carcinogens in cigarette smoke and smokeless tobacco are the tobacco specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'- nitrosonornicotine (NNN). These nitrosamines are present in mainstream and sidestream smoke at levels of 30-770 ng per cigarette and in smokeless tobacco products at levels of 100-89,000 ppb. NNK induces tumors of the lung, trachea and nasal cavity in Syrian golden hamsters; the lung, nasal cavity, liver an pancreas of F344 rats; and the lung in A.J mice. A mixture of NNN and NNK induced oral cavity tumors in F344 rats. The organospecificity of these compounds for tumor induction in rodents correlates with the incidence of tumors in humans. If a strategy for prevention of tobacco related cancers is to be developed a detailed understanding of the mode of action of tobacco carcinogens is needed. The metabolic activation of NNK occurs by alpha-carbon hydroxylation following one of two pathways. The first leads to the formation of methylating agent, the second to the formation of a pyridyloxobutylating agent. We will develop and validate assays to measure the metabolic activation of NNK and NNN. While a number of markers, such as nicotine levels, have been developed for monitoring exposure to tobacco they do not reflect metabolic activation of NNK or NNN. A sensitive GC-NICI-MS method for an NNK-DNA adduct has been developed and will be validated. In addition, an immunoassay for NNK-hemoglobin adducts will be developed and validated. These assays will be used to test human samples for NNK and NNN adducts of DNA and hemoglobin. The availability of these assays will allow us to determine if a correlation exists between DNA and hemoglobin adducts. This information is necessary for human screening programs since hemoglobin adducts are easier to measure but DNA adducts are biologically more important. This information will allow us to develop quantitative measures of risk which are more accurate than that possible using simple measures of exposure.